A detailed design of stable-isotope techniques, NMR spectroscopy and mass spectrometry, for studying drug-biochemical receptor interactions in vitro is proposed in this project. The main emphasis is on unraveling the bioorganic mechanism of actions of alkylating agents in terms of their reactions with RNA and DNA. The agents include methyl methanesulfonate, dimethyl sulfate, 1-methyl-3-nitro-1-nitrosoguanidine, 1-methyl-1-nitrosourea and their ethyl analogs, dimethylnitrosamine (activated by microsomal enzymes), alpha-acetoxydimethylnitrosamine, ethylene oxide, aziridine, 2-chloroethylamine, bis-(2-chloroethyl)amine, bis-(2-chloroethyl)methylamine, 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 2-chloroethyl isocyanate, cyclohexyl isocyanate. The reactions of both natural abundance and 13C-and 2H-enriched alkylating agents with mononucleotides, dinucleotides, RNA and DNA will be carried out in aqueous solution and maintained at neutrality by a pH-Stat, and the products will be unequivocally characterized by UV, IR, NMR and mass spectral analysis. The conformational variations will be determined by 1H-1H, 13C-1H, 31P-1H and 13C-31P coupling analysis. The relative chemical kinetics will be investigated by NMR using stable-isotope enriched agents. The alkylated product distribution will be calculated from NMR integration curves, and mass spectrometry using either GC-MS method or internal reference method. The reactive sites, relative chemical kinetics, conformational variations, cytotoxicity of alkylated products and product distribution will be utilized to correlate the structure-activity relationship.